Project Summary Emerging evidence shows that recurrence of the acute leukemia results from the activity of leukemia stem cells (LSCs). Leukemic stem cells represent a rare population of cells, which are capable of self-renewal, proliferation and differentiation into malignant blasts. LSCs are much more resistant to chemotherapy and radiation when compared to progenitors or blasts and new pharmacological agents targeting LSCs are urgently needed. Polycomb Repressive Complex 1 (PRC1) is a histone H2A E3 ubiquitin ligase and has a well- established role in the regulation of differentiation and maintenance of stem cell populations. PRC1 catalyzes the monoubiquitylation of lysine 119 at histone H2A (H2AK119ub) through heterodimeric Ring1B-Bmi1 E3 ligase. In this project we propose to develop small molecule inhibitors of the Ring1B-Bmi1 E3 ligase as new pharmacologic agents targeting leukemia stem cells. Targeting the Ring E3 ligases with small molecules is a very challenging task due to the lack of well-defined substrate binding pockets. We identified small molecules that bind to Ring1B-Bmi1 by employing fragment- based screening using NMR spectroscopy. We then performed extensive medicinal chemistry optimization of these compounds and very substantially improved their binding affinity, resulting in low micromolar inhibitors of the H2A ubiquitination in cells. Treatment of model LSC cell lines with Ring1B-Bmi1 inhibitors blocks colony formation and induces differentiation of these cells. In this project we hypothesize that potent Ring1B-Bmi1 inhibitors will impair self-renewal of leukemia stem cells and will block development of leukemia in vivo. Towards this goal, we propose to develop more potent inhibitors of the Ring1B-Bmi1 E3 ligase activity with optimized drug-like properties suitable for in vivo studies. We will employ medicinal chemistry and structure- based design approach to optimize Ring1B-Bmi1 inhibitors. We will extensively evaluate the mechanism of action of the Ring1B-Bmi1 inhibitors in the model LSC cell lines and in the primary patient samples. The optimized compounds will be assessed in vivo for their potential to block development of leukemia. Our studies will explore a new approach to target leukemia stem cells and may lead to development of highly valuable chemical probe compounds or novel pharmacologic agents for acute leukemia.